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The Journal of Experimental Biology 213, 210-224 Published by The Company of Biologists 2010 doi:10.1242/jeb.031781

Proteomic and physiological responses of leopard sharks (Triakis semifasciata) to salinity change

W. W. Dowd1,2,*, B. N. Harris3, J. J. Cech, Jr2 and D. Kültz1 1Physiological Genomics Group, Department of Animal Science and 2Department of Wildlife, Fish, and Conservation Biology, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, USA and 3Department of Biology, 3386 Spieth Hall, University of California, Riverside, CA 92521, USA *Author for correspondence at present address: Hopkins Marine Station of Stanford University, Pacific Grove, CA 93950, USA ([email protected])

Accepted 14 September 2009

SUMMARY Partially euryhaline elasmobranchs may tolerate physiologically challenging, variable salinity conditions in estuaries as a trade- off to reduce predation risk or to gain access to abundant food resources. To further understand these trade-offs and to evaluate the underlying mechanisms, we examined the responses of juvenile leopard sharks to salinity changes using a suite of measurements at multiple organizational levels: gill and rectal gland proteomes (using 2-D gel electrophoresis and tandem mass spectrometry), tissue biochemistry (Na+/K+-ATPase, caspase 3/7 and chymotrypsin-like proteasome activities), organismal physiology (hematology, plasma composition, muscle moisture) and individual behavior. Our proteomics results reveal coordinated molecular responses to low salinity – several of which are common to both rectal gland and gill – including changes in amino acid and inositol (i.e. osmolyte) metabolism, energy metabolism and proteins related to transcription, translation and protein degradation. Overall, leopard sharks employ a strategy of maintaining plasma urea, ion concentrations and Na+/K+-ATPase activities in the short-term, possibly because they rarely spend extended periods in low salinity conditions in the wild, but the sharks osmoconform to the surrounding conditions by 3 weeks. We found no evidence of apoptosis at the time points tested, while both tissues exhibited proteomic changes related to the cytoskeleton, suggesting that leopard sharks remodel existing osmoregulatory epithelial cells and activate physiological acclimatory responses to solve the problems posed by low salinity exposure. The behavioral measurements reveal increased activity in the lowest salinity in the short-term, while activity decreased in the lowest salinity in the long-term. Our data suggest that physiological/behavioral trade-offs are involved in using estuarine habitats, and pathway modeling implicates tumor necrosis factor  (TNF) as a key node of the elasmobranch hyposmotic response network. Supplementary material available online at http://jeb.biologists.org/cgi/content/full/213/2/210/DC1 Key words: elasmobranch, osmoregulation, urea, Na+/K+-ATPase, focal animal sampling, proteomics, MALDI-TOF-TOF mass spectrometry, pathway analysis, tumor necrosis factor.

INTRODUCTION need to determine whether (i) the animals are simply tolerating Salinity change is an important environmental challenge faced by challenging salinity conditions in exchange for greater net benefits, estuarine and coastal fishes, which have evolved a suite of (ii) they are capable of coping physiologically with those conditions, morphological, physiological and behavioral characteristics for or (iii) they employ other options such as behavioral avoidance (e.g. coping with such environmental variability. Species of the subclass moving away from low/changing salinity areas of the estuary) or Elasmobranchii (the sharks, skates and rays) commonly utilize behavioral modulation (e.g. reducing activity to conserve energy coastal and estuarine habitats, particularly at certain times of the for physiological acclimation when avoidance is not possible) to year or specific life-history stages (e.g. juvenile nursery areas) compensate for physiological ‘shortcomings’. Such diverse (Heupel et al., 2007; Springer, 1967). However, with relatively few possibilities highlight the need for an integrative approach to exceptions, most elasmobranchs exhibit low tolerance of salinity answer questions in ecological physiology, incorporating techniques change – the majority being stenohaline (some in freshwater) or that provide insight into responses at different levels of biological partially euryhaline. From a physiological perspective, partially organization (i.e. from individual cells to whole organisms). euryhaline elasmobranchs entering estuaries with variable salinity At the molecular level, we have previously applied 2-D gel may incur significant costs due to their urea-based osmoregulatory electrophoresis and mass spectrometry-based proteomics techniques strategy, which creates slightly hyperosmotic or isosmotic conditions to understand the adjustments made in fish tissues to physiological within the shark relative to full-strength seawater (SW) (Evans et challenges (Dowd et al., 2008; Kültz et al., 2007). Proteins are the al., 2004; Hazon et al., 2003). From an ecological perspective, primary executioners of cell functions, and the proteome integrates elasmobranchs in estuaries, particularly juveniles, may enjoy the changes in gene expression, mRNA stability and protein post- benefits of abundant food resources as well as spatial refuge from translational modification and turnover in response to environmental larger predators found in the oceans (Branstetter, 1990; Heupel et change (Kültz, 2005). Gel-based proteomics, like cDNA microarrays al., 2007). In order to better understand the potential trade-offs and other ‘omics’ approaches, is a powerful tool for generating new involved in using such dynamic, potentially stressful habitats, we mechanistic hypotheses that then can be tested by other means (e.g.

THE JOURNAL OF EXPERIMENTAL BIOLOGY Leopard shark proteomics 211

Matey et al., 2009). However, very few studies have attempted to for 2–62 days prior to the beginning of an experiment (2005: integrate proteomics with other levels of biological organization in 33.4±0.1 p.p.t., 13.5±0.2°C, 7.31±0.13 mg l–1 dissolved oxygen; ecologically relevant scenarios for ‘non-model’ organisms. Due to 2007: 33.8±0.1 p.p.t., 10.7±0.2°C, 7.69±0.07 mg l–1). Sharks were the unique osmoregulatory strategy of elasmobranchs and their basal fed ad libitum rations of squid (Loligo spp.) and/or Bay ghost shrimp position in the vertebrate phylogeny, unraveling the key players in (Neotrypaea californiensis Dana 1854) in the holding tank. their osmoregulatory response mechanisms will provide both Salinity experiments were conducted in flow-through evolutionary and ecological insights. 1600–1900 liter fiberglass tanks by varying the ratio of inflowing In this study, we exposed leopard sharks (Triakis semifasciata seawater (SW; ~34 p.p.t.) to freshwater (FW; 2.9 p.p.t.) to each tank Girard 1855) to salinity changes representative of those likely to be using two in-line flow meters. The water turnover rate in the encountered in estuaries in order to assess their responses from the experimental tanks was 32–45% per hour (h). Salinity was reduced molecular level through to the organismal level. Sharks provide gradually from full-strength SW (experiments 1 and 2 over 18 h; abundant tissue for proteomics and biochemistry, and they are experiment 3 over 11 h) in order to mimic rates of salinity change amenable to complementary techniques at the organismal level. likely to be experienced by sharks in the wild. To avoid stress from Leopard sharks inhabit California estuaries at all times of year, where aerial exposure, all movements between tanks were performed using they can experience salinity changes on several time scales (Fig. 1), a watertight sling. All procedures were approved by the University including stochastic changes due to extreme rainfall events or of California, Davis IACUC (Protocols 11745 and 12746). anthropogenic activities. These salinity changes can be fairly rapid, leading to shifts of as much as 18 parts per thousand (p.p.t.) in just Experiment 1: Short-term (48 h) osmoregulatory responses to 12 h. Leopard sharks have been captured in San Francisco Bay at salinity change salinities as low as 14 p.p.t. but they are more commonly encountered Sharks in this experiment were subjected to short-term (~48 h) at salinities greater than 18 p.p.t. (J. Diesel, Marine Science Institute, exposures to one of three salinity treatments: 33.3 p.p.t. (100% SW personal communication). These data suggest a lower physiological control), 27.6 p.p.t. (80% SW) or 20.7 p.p.t. (60% SW) [N18; tolerance threshold (i.e. leopard sharks are partially euryhaline) 1245±101 g; 68.5±2.1 cm total length (TL)]. Prior to the experiment, and/or behavioral avoidance of lower salinities in the wild. 12 of these sharks were anesthetized in FinquelTM MS-222 (Argent Because responses to environmental challenges are likely to vary Chemical Laboratories, Redmond, WA, USA) and cannulated via among tissues, depending on both their exposure to surrounding the caudal artery with a 1.5 m length of PE50 tubing using a 17G conditions and their functions, we assessed salinity-dependent Touhy needle to allow repeated blood sampling during the changes in both the rectal gland and gill proteomes. The mechanisms experiment (Hopkins and Cech, 1995). The cannula was filled with of salt secretion in the elasmobranch rectal gland have been heparinized elasmobranch Ringer solution (Anderson et al., 2002). thoroughly described (reviewed in Evans et al., 2004), yet we know During this short procedure each shark was weighed, sexed, little about how these functions are modulated by environmental measured and photographed for identification. Sharks were allowed change or how other aspects of the cellular response are integrated to recover in 100% SW in the experimental tanks for 44–68 h prior with higher levels of organization. The elasmobranch gill (the to salinity change. Sham surgeries were conducted on the remaining principal semi-permeable interface between water of changing six sharks; the procedure was identical to that for cannulated animals, salinity and the internal milieu) is thought to play a relatively limited except the cannula was never inserted. The sharks were not fed role in seawater osmoregulatory processes aside from acting as a during the recovery period or during the experiment. barrier to urea loss [but see Evans et al. (Evans et al., 2004) for a At the start of the experiment (time0 h), an initial blood sample review of the gill’s putative function in freshwater elasmobranchs]. (~0.6–0.7 ml) was drawn through the cannula while all sharks Using proteomics and physiological data in concert with laboratory remained at 100% SW. Salinity change commenced 6 h thereafter, measurements of behavior, we discuss mechanisms and propose and repeated blood samples were collected at 12 h (during salinity novel key regulators in the elasmobranch osmoregulatory response change), 24 h (immediately after salinity change) and 48 h. Water network. These mechanisms may underlie the trade-offs that samples were collected at each time point for comparison with the influence estuarine habitat use. blood plasma. Sharks were euthanized in ice-cold seawater by pithing at 53–54 h and were immediately weighed and measured. MATERIALS AND METHODS Animal collection, maintenance and salinity control Experiment 2: Long-term (3 weeks) osmoregulatory and Experiments were carried out at the Bodega Marine Laboratory, tissue responses to salinity change CA, USA, during June–August 2005 (experiments 1 and 2) and May The sharks in this experiment (N9; 2241±526 g, 81.5±5.5 cm TL) 2007 (experiment 3). Leopard sharks (N45) were collected in were exposed to the same salinity treatments (100%, 80% and 60% Bodega Harbor using a beach seine and transported in coolers SW) and rates of salinity change as those in experiment 1, but they containing oxygenated seawater to the laboratory. There they were remained at the final salinity for 3 weeks. Prior to the experiment, held in a 3.7 m diameter, flow-through tank at natural photoperiod each shark was lightly anesthetized in MS-222 and then weighed,

40 L01 30

20 Fig. 1. Long-term salinity record for a single monitoring station in Elkhorn Slough, a California estuary inhabited by leopard 10 sharks, demonstrating interannual, seasonal, and shorter scale Sa linity (P S U) (e.g. tidal) salinity variability. Data obtained from the Monterey 0 Bay Aquarium Research Institute’s Land Ocean 13 January 2004 16 February 2005 24 March 2006 Biogeochemical Observatory database (www.mbari.org/lobo/).

THE JOURNAL OF EXPERIMENTAL BIOLOGY 212 W. W. Dowd and others sexed, measured and photographed for identification. The sharks et al. (Dowd et al., 2008). Based on the results of our physiological were then allowed 48 h to recover from handling in 100% SW before measurements in experiment 1, we selected the 24 h time point as commencing the salinity change. These sharks were not cannulated, being representative of the most physiologically challenging (i.e. because the cannulae tend to clot within 3–4 days. The sharks were largest osmotic gradient between shark and environment) conditions fed a controlled daily ration of squid (~2% body mass per day). faced during the short-term experiment. Proteomics analyses were At the end of the experiment a blood sample was drawn from carried out on tissues from the same individuals used in experiment the caudal artery, and sharks were then euthanized as in experiment 3 (N5 or 6 per treatment for rectal gland; N3 for gill for 50% and 1. In order to clear the body of background red blood cells (RBC) 100% SW), with separate gels run for each individual and each for biochemistry and proteomics, we perfused the animal via the tissue. Briefly, proteins were extracted, precipitated in acetone with heart with elasmobranch Ringer solution and the sinus venosus was 10% trichloroacetic acid, loaded (1 mg per sample) onto 18 cm severed to allow exsanguination for several minutes after the gills immobilized pH 3–10 non-linear gradient strips by passive overnight turned white. Following perfusion, we collected gill (second left rehydration, separated in the first dimension by isoelectric focusing, arch), rectal gland and kidney (just anterior to mid-point). Two equilibrated with dithiothreitol and iodoacetamide, and then run on replicates of each tissue were snap-frozen in liquid nitrogen and 16 cm second dimension SDS-PAGE gels (Dowd et al., 2008; then stored at –80°C until analysis. Valkova et al., 2005). 2-D gels were fixed and stained with Coomassie Brilliant Blue G250, destained in purified water and then Experiment 3: Short-term (24 h) osmoregulatory and tissue scanned for densitometry. The individual gel images were aligned, responses to salinity change fused into a master gel image for each tissue and quantified based In this experiment, 17 sharks (1668±130 g, 74.8±2.0 cm TL) were on the normalized volume of each spot (relative to the total volume exposed to gradual changes in salinity from 100% SW to 100% SW of all spots) using Delta 2D software (v3.6, Decodon, Germany) (control, N5), 75% SW (N6) and 50% SW (N6). After 24 h, the (Dowd et al., 2008). Protein spots whose abundance changed sharks were euthanized and perfused as above, and then blood and significantly [P<0.05; expression ratio >2 (increased by >100%) or tissues were collected in duplicate as in experiment 2. <0.5 (decreased by >50%)] between treatments were excised and processed for MS identification. Hematological, plasma and tissue analyses Hematology MS and bioinformatics database searches Heparinized blood samples were collected in microfuge tubes and Protein spots of interest were digested overnight with MS-grade stored on ice until analysis (<1 h). We determined hematocrit (HCT; trypsin, and the resulting peptide fragments were eluted in 60% % packed red blood cells) using heparinized hematocrit tubules. In acetonitrile:1% trifluoroacetic acid (TFA). The peptide solution was experiment 1 only, we determined RBC count (105 cells per mm3) then dried by vacuum centrifugation and resuspended in 1% TFA. using diluting pipettes and hemocytometers on a ϫ40 objective light The peptides were desalted and concentrated using C18 ZipTips microscope (Cooper and Morris, 1998). Using these data, we (Millipore, Billerica, MA, USA), spotted in 0.5 l increments on a calculated mean corpuscular volume (MCV). stainless steel MALDI target and overlaid with -cyano-4- hydroxycinnamic acid matrix (Dowd et al., 2008). Peptides were Plasma constituents analyzed on an Applied Biosystems 4700 Proteomics Analyzer Blood samples were centrifuged at 5000g for 5 min, and the plasma MALDI-TOF-TOF tandem mass spectrometer (Foster City, CA, was removed and frozen before analysis. Plasma samples were USA) in reflector positive mode (MS) with subsequent post-source analyzed in triplicate for Na+ and K+ concentrations (mmol l–1) by decay (PSD) and collision-induced dissociation (CID) tandem flame photometry (Instrumentation Laboratory 343, Bedford, MA, fragmentation (MS/MS) as previously reported (Dowd et al., 2008; USA), Cl– concentration (mmol l–1) using a chloride titrator Lee et al., 2006). Peak lists from the resulting MS and MS/MS (Radiometer CMT10, Copenhagen, Denmark; or Labconco Digital spectra were searched jointly against both the NCBI and SwissProt Chloridometer, Kansas City, MO, USA), and osmolality non-redundant databases using the Mascot search algorithm (Perkins (mOsmol kg–1) using a vapor pressure osmometer (Wescor VaproTM et al., 1999) with mass error tolerances of 50 p.p.m. for MS and 5520, Logan, UT, USA). Plasma urea concentration (mmol l–1) was 0.3 Da for MS/MS. The MS/MS spectra were also submitted for determined spectrophotometrically using a biological urea nitrogen automated de novo peptide sequencing analysis to both GPS kit (Randox Laboratories, Crumlin, UK) modified for use on a Explorer De Novo Explorer (Applied Biosystems) and PEAKS microplate reader. Total plasma protein concentration (g %) was Studio v4.5 software (Bioinformatics Solutions, Toronto, Canada) measured by refractometry. (Ma et al., 2003), again with mass error tolerances of 50 p.p.m. for MS and 0.3 Da for MS/MS. The resulting de novo amino acid Tissue measurements sequences for the tryptic peptides were then searched against the At the conclusion of all three experiments we collected 2–3 g of NCBI non-redundant database using MSBLASTP (Shevchenko et epaxial white muscle (ventral to the second dorsal fin) in duplicate. al., 2001) and the PEAKS proprietary search engine, respectively. White muscle tissues were then dried to constant mass at 55°C to This de novo sequencing approach has proven useful in identifying determine the percentage muscle moisture. In experiments 1 and 2 peptides from non-model organisms – including sharks – that do the rectal gland and liver were excised and weighed to assess the not match predicted peptide mass fingerprint peaks from proteins relationships of rectal gland mass and liver mass to body mass. in bioinformatics databases (Dowd et al., 2008; Lee et al., 2006). Consensus protein identifications were determined based on Mascot Proteomics (both NCBI and SwissProt), MSBLASTP and PEAKS scores. 2-D gel electrophoresis and image analysis Matches solely from de novo sequencing scores were only accepted Proteins regulated by exposure to salinity change in the leopard shark when more than 1 peptide matched the protein. When possible, rectal gland and gill were identified using 2-D gel electrophoresis matches were confirmed by agreement of molecular mass (Mr) and and tandem mass spectrometry (MS) following the methods of Dowd isoelectric point (pI) between the database match and the protein

THE JOURNAL OF EXPERIMENTAL BIOLOGY Leopard shark proteomics 213 spot of interest on 2-D gels. However, because many of the matched Behavioral sampling proteins were not sequenced in elasmobranchs, this is not always To quantify behavioral responses to salinity change in the laboratory, possible due to sequence differences and/or post-translational we conducted two types of behavioral sampling. During experiments modifications of proteins. 1 and 2, we used repeated, 7-minute focal animal sampling intervals (Altmann, 1974). Minutes 1 and 7 were devoted to counting tail- Pathway analysis beat frequency, a proxy for swimming speed (Lowe, 1996). Minutes The lists of changing proteins were analyzed separately for rectal 2–6 were divided into 20-second intervals, and the behavior of the gland and gill using Ingenuity Pathways Analysis (IPA) software shark in that interval (swimming or resting) was recorded. In (Ingenuity Systems®, www.ingenuity.com). Identifiers for the experiment 3, we also used a point-sampling approach in which the mouse or human homologs of the identified proteins were uploaded behavior of each shark (swimming or resting) was recorded every into IPA, and Gene Ontology (GO) annotations for molecular 4–6 h throughout the experiment. Data collected using both of these functions were generated from the manually curated Ingenuity sampling techniques were analyzed as the proportion of time spent knowledge base. IPA Canonical Pathways Analysis identified the in each behavioral state. In experiment 2 we also counted the number cellular pathways that were significantly represented in the dataset of sharks swimming 3–4 times per day. In all experiments, based on a right-tailed Fisher’s exact test. The protein identifiers individuals were distinguished using the unique spot patterns on the were then overlaid onto a global molecular interaction network dorsal side of the head. For the short-term experiments 1 and 3, the developed from the primary literature and contained in the data were collected as salinity was changing. Ingenuity knowledge base. Networks containing the salinity- regulated genes were then algorithmically generated based on their Statistical analyses connectivity, with each network assigned a statistical likelihood In most cases, treatment groups were compared using one- or two- score (Calvano et al., 2005). way analysis of variance (ANOVA) with salinity and/or time as factors as appropriate, and post hoc multiple comparisons were Biochemical assays carried out using Tukey’s Honestly Significant Difference test. Na+/K+-ATPase activity Behavioral activity data based on proportions were first arcsine The activity of Na+/K+-ATPase in gill, rectal gland and kidney at square root transformed. For plasma constituents, we analyzed all 25°C was determined following the method of McCormick data from experiments 1 and 2 together using a mixed-effect model (McCormick, 1993), as previously applied to elasmobranchs with salinity and time as factors in SAS 9.1.3 (SAS Institute, Cary, (Piermarini and Evans, 2000). NC, USA); we included shark ID in the model to account for the repeated sampling structure in experiment 1. The relationships of Caspase 3/7 activity liver mass and rectal gland mass with salinity were assessed using Apoptosis-related pathways might be influenced by salinity change analysis of covariance (ANCOVA) with body mass as the covariate. in leopard shark tissues, as they are in teleost gill (Kammerer and Significance level in all cases was set at 0.05. Normalized protein Kültz, 2009). Thus, we measured the activity of caspases 3 and 7, expression levels of the experimental 2-D gels in the 50% and 75% two proteases that are early effectors of apoptosis, using a SW treatments were compared with control 100% SW gels using luminescent assay (Caspase GloTM 3/7 assay kit, Promega, Madison, a t-test in Delta 2D software (Dowd et al., 2008). All other statistical WI, USA) modified for whole-tissue homogenates (Kammerer and analyses were performed in Statistica© 6.1 (StatSoft, Tulsa, OK, Kültz, 2009). Tissues were homogenized in a lysis buffer USA). (10 mmol l–1 Tris-HCl pH 7.5, 100 mmol l–1 NaCl, 0.1 mmol l–1 EDTA, 0.2% Triton-X100), and caspase activity was normalized to RESULTS total protein, as determined by a bicinchoninic acid (BCA) assay Hematological, plasma and tissue analyses (Pierce). Hematology There were no differences in HCT among the salinity treatments Chymotrypsin-like proteasome activity (Table 1); the two-way ANOVA indicated an effect of time We measured chymotrypsin-like proteasome activity in gill using (P0.044) but no effect of salinity (P0.152) or interaction a luminescent assay (Proteasome-GloTM Chymotrypsin-Like Cell- (P0.190). The data from 48 h also indicated no effect of salinity Based Assay, Promega). Tissues for this assay were homogenized on RBC count (P0.627) or MCV (P0.175) (Table 1). in a hypotonic buffer (10 mmol l–1 Tris-HCl pH 7.5, 5 mmol l–1 –1 –1 MgCl2, 0.5 mmol l DTT, 5 mmol l ATP) per the manufacturer’s Plasma constituents recommendation. Proteasome activity was normalized to total Leopard shark plasma remained iso- or hyperosmotic to the SW protein based on a BCA assay. medium in all conditions, but plasma osmolality decreased with

Table 1. Hematological variables [hematocrit (HCT, %), mean corpuscular volume (MCV, m3), and red blood cell count (RBC, cells mm–3)] for leopard sharks exposed to 50–60%, 75–80% or 100% seawater (SW) for 24 h, 48 h and 3 weeks Time 50–60% SW N 75–80% SW N 100% SW N 24 h HCT 19.3±0.7a,b 6 16.6±1.5a,b 6 22.7±2.3a 5 48 h HCT 16.9±1.1a,b 4 16.6±0.5a,b 3 15.7±1.8b 4 MCV 0.00043±0.00004 0.00044±0.00001 0.00032±0.00003 RBC 408,000±43,000 371,000±31,000 487,000±119,000 3 weeks HCT 20.2±0.8a,b 3 17.0±1.3a,b 3 19.0±0.5a,b 3 Values are means ± s.e.m. Different lowercase superscripts for HCT indicate statistically different groups, as determined by a two-way ANOVA with time and salinity as factors. There were no differences in MCV or RBC among treatments at 48 h.

THE JOURNAL OF EXPERIMENTAL BIOLOGY 214 W. W. Dowd and others salinity to different degrees in the 60% and 80% SW treatments levels up to 48 h in the 80% SW treatment, but it also declined at and continued dropping over the 3 weeks (Psal<0.001, Ptime<0.001, 3 weeks (Fig. 2E). Leopard sharks maintained their plasma Psalϫtime<0.001) (Fig. 2A–C). The osmotic gradient between the hypoionic to the SW medium. Plasma sodium dropped with shark and the water was greatest in the 60% treatment at 24 h, decreasing salinity to different degrees in the 60% and 80% SW while the gradient at 3 weeks was similar in magnitude to that at treatments (Fig. 2G–I) but it stabilized by 48 h in each treatment time 0 h (Fig. 2A). Urea (Psal<0.001, Ptime<0.001, Psalϫtime0.065) (Psal<0.001, Ptime<0.001, Psalϫtime<0.001). Notably, in the 60% SW decreased slightly with decreasing salinity in the 60% SW treatment at 24 h plasma sodium is almost equal to that of the water treatment over 48 h, and subsequently exhibited a dramatic drop but the sharks re-established a (smaller) hypoionic gradient by 48 h. at 3 weeks (Fig. 2D). Similarly, urea fluctuated around control Chloride decreased with decreasing salinity in the 60% SW

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600 16 Fig. 2. Leopard shark plasma (l) and seawater (SW) (l) osmolality 14 (A–C) and concentrations of urea (D–F), sodium ions (G–I), 500 12 chloride ions (J–L), and potassium ions (M–O) in 60% SW (top), 400 10 80% SW (middle) and 100% SW (bottom) treatments. Time points 8 0 h, 12 h, 24 h and 48 h were repeated samples from cannulated 300 a a 6 a,b a sharks in experiment 1; the 3 week samples were obtained from a a,b a,b b,c 4 different set of animals in experiment 2 by caudal puncture. )

a,b,c ) a,b 200 c Different lowercase letters represent statistically different groups

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–1 2 M 600 16 across all times and treatments for that variable. 14 500 12 400 10 a tion (mmol l a tion (mmol l 8 a,b a,b a,b 300 a,b a,b a,b,c 6 a,b a,b a,b 4 a,b 200 K 2

concentr N concentr – 600 + 16 K Cl 14 500 12 400 10 8 a a a a 300 a,b 6 a,b a,b a,b 4 b 200 L 2 O a,b s s 0 h 24 h 48 h 0 h 24 h 48 h 3 week 3 week Time

THE JOURNAL OF EXPERIMENTAL BIOLOGY Leopard shark proteomics 215 treatment but not in the 80% SW treatment (Psal<0.001, Proteomics Ptime<0.001, Psalϫtime0.008) (Fig. 2J–L). However, for unknown Rectal gland reasons the 0 h mean chloride concentration for 80% SW was The master gel image for rectal gland contained 588 protein spots significantly lower (257±11 mmol l–1) than for the 60% and 100% that were quantified (Fig. 4). The abundance of 11 spots changed SW treatments (273±10 and 274±4 mmol l–1, respectively). Plasma significantly in the 75% SW treatment relative to 100% SW potassium concentrations were more tightly regulated across controls (7 decreased, 4 increased), and 22 spots changed abundance salinity treatments (Psal0.052), although there was a small effect significantly in the 50% SW treatment relative to 100% SW of time (Ptime0.0191, Psalϫtime0.147) (Fig. 2M–O). Plasma controls (16 decreased, 6 increased) (Table 2). Of the 28 total protein protein content decreased with salinity at both the 48 h and 3 week spots regulated by salinity change, 20 (71%) were identified using time points (Psal<0.001, Ptime0.811, Psalϫtime0.357) (Fig. 3A), MS combined with bioinformatics searches (Table 2). Other protein suggesting hemodilution. spots yielded high quality mass spectra that did not produce matches in any of the database searches. Tissue analyses Based on GO annotations and a manual review of the literature, The 50–60% SW and 75–80% SW treatments gained muscle we categorized the identified proteins according to their likely moisture, and it stabilized by 48 h (Psal<0.001, Ptime0.003, functions (Table 2). Several proteins related to energy (i.e. ATP) Psalϫtime0.202) (Fig. 3B). Animals also gained weight in the production (glycolysis, TCA cycle, malate aspartate shuttle) 50–60% SW (9.0±1.2% body mass) and 75–80% SW decreased in abundance. Seven spots were identified as the (1.9±0.8% body mass) treatments in the 48 h experiment despite mitochondrial ATP synthase alpha subunit, two of which increased not feeding, further supporting water influx. There was no in abundance in 50% SW while four decreased (Table 2). This effect of salinity on liver mass (ANCOVA, P0.793). phenomenon is common in 2-D gel studies (e.g. Dowd et al., 2008), Meanwhile, rectal gland mass tended to decrease slightly in and it suggests post-translational modifications such as sharks exposed to lower salinities for both the 48 h and 3 week (de)phosphorylation of the corresponding proteins. Overall, this treatments (ANCOVA, P<0.001). protein expression pattern suggests a decrease in oxidative phosphorylation at 50% salinity in rectal gland, consistent with other aspects of energy metabolism. There were also consistent decreases in heterogeneous nuclear ribonucleoproteins and PRP4 pre-mRNA processing factor, all related to transcription and mRNA processing. 6 Three keratins involved in the stability of the cytoskeleton also A c c decreased. By contrast, creatine kinase and methylmalonate- 5 semialdehyde dehydrogenase both increased in 50% SW. Five b protein spots were consistently up- or downregulated in both 4 + a,b treatments (Table 2); two of them were identified as H -transporting a ATP synthase alpha subunit (increased both treatments) and 3

2 Pl as m a protein (g % ) 1 100- 48 hours 75- 3 weeks 0 60 80 100 50- 82 B a a,b 37- 81 O) 2 a,b,c 80 a,b,c,e c,d,e 25- b,c,e 79 Molec u l a r m ass (kD ) 20- d,e 78 d,e d

M us cle moi s t u re ( % H 77 24 hours 48 hours 3 weeks 76 3.5 4 5 6 7 8 9 9.5 50–60 75–80 100 pI Salinity (% SW) Fig. 4. Master 2-D gel for leopard shark rectal gland at 24 h, formed by warping and fusing 16 individual gel images together in Delta 2D software. Fig. 3. Plasma protein concentration (A) and muscle moisture content (B) Protein mixtures were separated by isoelectric point (pI) in the horizontal for leopard sharks exposed to salinity changes in experiments 1 (48 h), 2 (3 dimension and molecular mass in the vertical dimension. Outlines weeks) and 3 (24 h). Different lowercase letters represent statistically represent the 588 protein spots analyzed across the three salinity different groups, as determined by two-way ANOVA with time and salinity treatments, and red labels indicate spots that were significantly up- or as factors. Due to technical failures only one data point was obtained at downregulated in 50% and/or 75% seawater (SW) relative to 100% SW 80% SW for 48 h; this time point was excluded from the statistical analysis. controls.

THE JOURNAL OF EXPERIMENTAL BIOLOGY 216 W. W. Dowd and others 3 4 7 .9 . . gel 1 7 r 49.5 3 2.5 3 7.5 3 7.5 38 .1 3 2. 3 7.4 29.6 3 1. 8 2 1 (kD a ) M 8 833 9 2 1 .2 16.7 . . . . pI 5.9 7.5 7.7 5.2 5 3 .4 gel 4 7.2 5 3 . 8 6 6 (pH) 8 8 8 r 1.9 6 4.1 5.9 26.2 – – – – – 4.1 5.5 9.4 5 69.1 M 3 6. 16.9 6 22. 3 9.5 5.2 50 3 9.6 5.53 9.6 2 3 .1 5.5 2 3 3 4.4 5 20.6 3 6. 22.1 5.5 21.2 theor (kD a ) 83 3 33 33 83 8 – – – – – pI 8 .5 (pH) theor – – – – – – 8 ( 3 )5. 8 2 ( 3 ) 9 27.4 5.4 1 8 .6 ss ion r a tio (<0.5 or >2.0) criteri for 3 )7. – – – – 5.5 17 – – – – – S DCID 8 (2) 3 ) 99 (7) – 5.2 46. –7 – – – – – – <0.05) a nd expre 8 (2) 9 3 ( )9 109 (2) – – 7. 151 ( 3 ) – – 10 117 7 27 M S BLA TP PEAK S S wi ss Prot a nd NCBI non-red u nt s e qu ence d t abas . 29 3 3 – – – – – 3 2 190 (4) 99 (5) 99 (6) 9.1 10 4. 12 1 8 2 ( 3 ) 91 ( 3 ) 91 (2) 6.9 3 5 –25 90 ( 3 ) 96 ( 3 )9.4 247 (4)25 99 (4) 95 (4) 9.5 – 5 3 ( )– 9. 33 38 – – 107 (2) – – 9.2 – –– – – – 6.1 50. – – – – –– 3 7 16 – 72 ( 1 3 62 3 0 – – – – – M as cot de novo s e qu enced peptide from M S /M pectr a th t m tched the protein in 3 4420 3 015 29 99 24 – 94 (4) 77 (4) 9.5 16 1 8 22 22 206 22 11 3 (2) 99 (7) 94 (4) 9.2 59. 88 10 – – – 69 (2) 74 (2) 8 8 1 – – – – – 8 1221 69 1 8 4 33 3 79 3 7 8 P S D Cov. CID Cov. P S DP ) a re pre s ented for b oth the top d t abas e hit (theoretic l, theor.) nd from leop rd h rk r 3 2046 1 3 42 8 7 92 7 3 ––––– – – – – – – – – – – 5.5 20. 3 – – – – – – – – – – 4.6 21.1 No. 383 1 38 21 8 2190152 215 44 – – 167 ( | tche s . The n u m b er of i Acce ss ion gi|2909610 76 14 – – 24 3 (5) 96 (4) – 6.1 50. gi|2909610 101 2 3 gi|1 3 50 8 22 gi|2 gi|2 8 6 3 0226 206 47 157 57 – 99 (6) 42 ( 3 )7. gi|2 8 6 3 0 33 4 156 22gi|2 8 6 3 0 33 219 210 gi|2 8 6 3 0 33 2 254 26gi|2 8 6 3 0 33 4 1 83 gi| 3 026 8 616 125 47 – – – – – 6. gi|6 8 161102 – – 125 17 – – – 6.5 26.1 5.4 24.6 gi|47207 3 17 gi|2 8 6 3 0226 150 20 gi|5 88 0124 8 gi|14 8 22 3 147 gi|11 8 562406 160 gi|1091 gi|1164 ) ) ) ) ) ) (MDH1) gi|292427 8 7 111388 2 Macaca ) (ATP5A1) ) (ATP5A1) a re s hown for M as cot m Mus musculus 3 ( Mus musculus ) (KRT1 8 )g ) (PGAM2) ) (KRT 8 ) (KRT 8 ) (CKM) ) (PRPF4) , mitochondri a l ( Ictalurus punctatus – –– – –– –– – –– –– + cleoprotein A1 ( % s e a w ter ( S W)] nd m ass pectrometry (M ) identific tion core for leop rd h rk rect l gl protein reg u ted b y sa linity ch nge u cle a r ri b on cleoprotein A u cle a r ri b on Protein identific a tion ( abb revi tion) ) (ATP5A1) ) (ATP5A1) ) (ATP5A1) ) (ATP5A1) ) (HNRNPA1) ) (ATP5A1) laevis (GAPDH) proce ss ing f a ctor) ( Homo sapiens (IDH2) -tr a n s porting ATP ynth as e lph subu nit i oform 1 ( Scyliorhinus canicula -tr a n s porting ATP ynth as e lph subu nit i oform 1 ( Petromyzon marinus -tr a n s porting ATP ynth as e lph subu nit i oform 1 ( Petromyzon marinus -tr a n s porting ATP ynth as e lph subu nit i oform 1 ( Scyliorhinus canicula (HNRNPA 3 ) mulatta (ALDH6A1) (HNRNPA1) + + + + Mitochondri a l ATP s ynth as e, F1 complex, lph subu nit ( Xenopus Glycer a ldehyde- 3 -pho s ph te dehydrogen as e ( Triakis scyllium S erine/threonine-protein kin as e PRP4 homolog (PRP4 pre-mRNA Heterogeneo us n Pho s phoglycer a te m u t as e 2 ( Danio rerio Heterogeneo us n u cle a r ri b on cleoprotein A1 i s oform 1 ( H I s ocitr a te dehydrogen as e 2, NADP P 0.562 0.297 Type II ker a tin K 8 ( Scyliorhinus stellaris 0.641 Type II ker a tin K 8 ( Scyliorhinus stellaris 0.024 9 0.009 2 0.164 Cre a tine kin as e ( Scyliorhinus canicula 0. 8 0.7 0.107 0.47 0.124 H 0.94 0.747 Cyto s olic m a l te dehydrogen as e ( Acipenser brevirostrum 0. 3 9 0.102 H 0.67 0.265 ATP s ynth as e a lph subu nit ( Crocodylus siamensis 0.45 0.02 8 0.64 0.265 Ker a tin, type I K1 8 ( Scyliorhinus stellaris 0.59 0.152 Heterogeneo us n 0.65 0.094 1.24 0.522 H 0.29 0.0520. 3 ATP s ynth as e a lph subu nit ( Tetraodon nigroviridis 1.19 0.5 8 4 Methylm a lon te- s emi ldehyde dehydrogen as e ( Xenopus laevis 0.55 0.0 3 9 0. 3 1 0.044 0.54 0.07 8 3 . 8 9 0.027 0.6 3 0.42 0.029 0.29 0.06 3 4.0 8 0.76 0. 3 9 0.02 3 75 % P 0.01 0.019 0.041 0.942 0.011 0.046 0.1 3 7 Expre ss ion r a tio s 2. Expre ss ion r a tio s [rel tive to control 100 0. 3 0. 3

0.27 0.029 0.4 8 2. 3 1 0.027 0. 3 5 0.009 2.09 0.0 3 7 2.2 0.04 3 0.44 0.01 2. 8 4 0.02 2.62 0.017 0.45 0.027 0.4 8 2.09 0.049 0.25 0.02 8 0. 8 4 0.656 2.44 0.0 3 1 0.25 0.016 0.22 0.01 8 0.47 0.01 3 0.47 0.02 3 2.26 0.046 0. 3 2 0.01 0. 3 7 0.007 0.4 8 0.27 0.029 0.6 8 50 % a n ly s i in th t tre tment. D from b oth po t- o u rce dec y (P S D) nd colli ion-ind ced di ss oci tion (CID) M /M mode were e rched g the 3 4 2 1 7 9 5 6 8 12 15 14 11 17 3 1 1.0 3 21 10 1 8 22 2 3 3 0 0.5 0.1 33 16 20 19 29 0.44 0.096 1 3 3 2 1.65 0.152 33 T ab le S pot gel s (gel). M S BLA TP a nd PEAK s e rche re hown in p renthe fter the core . I oelectric point (pI) molec u l r m ass ( S e qu ence cover a ge for peptide m ass fingerprint s pectr (Cov.; % of over ll ence) M S (iv) Pho s phocre a tine circ u it (ix) Unidentified (i) Glycoly s i (iii) M a l te as p rt s h u ttle (v) Oxid a tive pho s phoryl tion (vi) Amino a cid met ab oli s m (vii) mRNA proce ss ing a nd tr n s port (viii) Cyto s keleton (ii) TCA cycle Protein s were gro u ped b y p t a tive f nction bas ed on Gene Ontology nnot tion nd m n ua l review of the liter re. V e in old font met oth ti tic ( P

THE JOURNAL OF EXPERIMENTAL BIOLOGY Leopard shark proteomics 217 heterogeneous nuclear ribonucleoprotein A1 (decreased both mannose metabolism (PMM2, ALDOA, P0.003), treatments). glycolysis/gluconeogenesis (ALDOA, GAPDH, P0.009), protein We focused on the pathway analysis results for the 50% SW ubiquitination pathway (PSMD12, PSMC2, PSMA2, P0.02) and treatment, because only three proteins mapped to a single network inositol metabolism (ALDOA, P0.02). The protein list mapped in the 75% SW treatment. This network was associated with to three IPA networks with functions associated with (i) DNA functions in DNA Replication, Recombination, and Repair; Post- Replication, Recombination, and Repair, Cancer, Cell Death Translational Modification; and Cancer (P10–8). The 20 identified (P10–45), (ii) Cell Cycle, Cell Death, Cell-mediated Immune proteins in the 50% SW list matched to 12 unique protein identifiers Response (P0.01), and (iii) Carbohydrate Metabolism, Nucleic in IPA. The top GO functions associated with these proteins were Acid Metabolism, Small Molecule Biochemistry (P0.01) (see (1) cellular assembly and organization (KRT8 and KRT18: Fig. S2 in supplementary material). stabilization, organization and quantity of keratin filaments, –7 P5.6ϫ10 ; CKM: aggregation of cellular membrane, P0.002), Biochemical assays (2) cell-to-cell signaling and interaction (KRT8 and KRT18: Na+/K+-ATPase activity –5 activation of fibroblast cell lines, P1.6ϫ10 ), (3) amino acid The activity of Na+/K+-ATPase exhibited different patterns for the –4 metabolism (ALDH6A1: catabolism of -alanine, P7.9ϫ10 , (4) three tissues assayed. Gill demonstrated consistently low activity carbohydrate metabolism (CKM: quantity and consumption of across all treatments and times (P 0.971, P 0.864) (Table 4). ϫ –4 ϫ –3 sal time glycogen, P7.9 10 ; PGAM2: flux of glucose, P5.5 10 ), (5) In the rectal gland, Na+/K+-ATPase activity in the 50% SW cellular compromise (KRT18: collapse of keratin filaments, treatment tended to decrease relative to the other treatments at 24 h P7.9ϫ10–4). The top canonical pathways associated with the (Psal0.011) (Table 4). Unfortunately, we did not have sufficient dataset were inositol metabolism (ALDH6A1, P0.01), glyoxylate tissue to assess changes in rectal gland activity at 3 weeks. Kidney and dicarboxylate metabolism (MDH1, P0.01), citrate cycle Na+/K+-ATPase activity increased in the lower salinity treatments, (MDH1, IDH2, P0.02), urea cycle and metabolism of amino groups doubling in 50–60% SW at 3 weeks (Psal<0.001, Ptime<0.001) (CKM, P 0.02), and -alanine metabolism (ALDH6A1, P 0.04).    (Table 4). The protein list mapped to one network with functions associated with Antigen Presentation, Antimicrobial Response, and Cell- –30 Caspase activity mediated Immune Response (P10 ) (see Fig. S1 in supplementary There were no significant effects of salinity on normalized caspase material). 3/7 activity in either gill (P0.706) or rectal gland (P0.197) at 24 h (Table 5). Overall, relative caspase activity was higher in gill, which Gill may reflect higher constitutive levels of cell turnover in this tissue The master gel image for gill contained 1037 protein spots that were than in rectal gland. quantified. Abundances of 70 spots changed significantly in the 50% SW treatment (45 decreased, 25 increased) relative to 100% SW Chymotrypsin-like proteasome activity controls (Table 3). Of these 70 spots, 26 (37%) were identified using There was no significant effect of salinity (P0.752) or time MS combined with bioinformatics searches (Table 3). (P 0.989) on gill chymotrypsin-like proteasome activity (Table5). The gill proteins were categorized in the same manner as for rectal  gland (Table 2). As for rectal gland, proteins involved in energy production (ALDOA in glycolysis), transcription/translation (UBTF, Behavioral sampling TOP1, EEF2) and the cytoskeleton (EZR, PLS3, ACTB) decreased In the short-term experiments, there was a significant effect of with decreasing salinity. There were fewer changes associated with salinity on swimming activity at 24 h (P0.046) (Fig. 5A), with the energy production in gill than in rectal gland. Notably, several 50–60% SW group spending a greater proportion of time swimming. components of the 26S proteasome (PSMD12, PSMC2, PSMA2) We found similar, but statistically insignificant, trends for swimming were identified in one or more spots that changed in abundance in activity (P0.285) and tail-beat frequency (P0.171) for 48 h. The gill, including pairs of protein spots with opposite changes in number of behavioral changes (blocks of time in which both rest expression that suggest post-translational modifications of and swim occurred) did decrease with decreasing salinity in the 48 h proteasome constituents [e.g. (de)phosphorylation] with salinity experiment (P0.016), suggesting that the sharks were swimming change. more consistently at lower salinities. In the 3 week treatment the The 26 identified gill proteins regulated by salinity change pattern of activity was reversed, with fewer sharks active in the 60% matched to 19 unique identifiers in IPA. The top GO functions SW group compared with the other treatments based on the point associated with these proteins were (1) amino acid metabolism sampling data (Kruskal–Wallis ANOVA: H2,4722.00, P<0.001). (DDAH1, OAT, PEPD: arginine and ornithine metabolism, Tail-beat frequency (P0.291) and swimming activity (P0.314) 5.3ϫ10–4; PMM2: glycosylation of amino acids, P0.05), (2) for 3 weeks exhibited similar, although insignificant, trends small molecule biochemistry (PMM2: biosynthesis of GDP-D- (Fig. 5B); discriminatory power at 3 weeks was limited by relatively mannose, P0.001; CPOX: biosynthesis of heme, P0.01; small sample sizes. DDAH1: biosynthesis of nitric oxide, P0.04; PPA1: formation of ADP-D-ribose, P0.001), (3) DNA replication, recombination DISCUSSION and repair (TOP1, UBTF: conformational modification of DNA, Salinity change has profound effects on leopard sharks at multiple P0.001), (4) carbohydrate metabolism (PMM2: biosynthesis of levels of organization, from proteins to behavior. The integration GDP-D-mannose, P0.001; PPA1: formation of ADP-D-ribose, of proteomics with traditional physiological measurements and P0.001; GAPDH: quantity of UDP-N-acetylglucosamine, behavioral data for a species with unremarkable salinity tolerance P0.006), and (5) cell cycle (TOP1: organization of nucleoli, allows us to describe some of the potential trade-offs inherent in P0.003; GAPDH: length of telomeres, P0.04). The top the use of estuaries by partially euryhaline elasmobranchs and canonical pathways associated with the dataset were fructose and suggests novel paths for future research.

THE JOURNAL OF EXPERIMENTAL BIOLOGY 218 W. W. Dowd and others 3 r 0. 8 8 . 3 9 22 M gel 52. 3 3 3 9. 3 1. 8 3 2.6 3 9. 3 9.4 26.7 (kD a ) 2. 62 pI 6.7 79.1 5.9 2 8 . gel (pH) r .6 6 27.6 8 8 .7 6.1 26.4 1.9 6 1.9 6.1 3 15.7 M 6 8 .6 5.9 60.9 91.4 6.1 5 8 .1 3 6.2 5.9 19.7 50.9 6.1 20. 96.5 4.9 77 33 . 3 33 . 3 theor (kD a ) 33 8 4 46.2 6 8 5496 u mn s as in T ab le pI 8 .2 6. 3 6.1 16.7 6. 3 (pH) b y sa linity ch a nge theor – 8 5 (2) 5. 333 88 (4) 6. 33 s reg u l a ted (5) 4 3 (2) S DCID – 8 (5) 96 ( 3 ) 9.6 2 3 .9 5.6 24.7 3 (2) – 7.7 1 8 .4 4. 8 3 (4) 92 (5) 9.6 2 3 .9 5.7 24.7 83 88 (5) u ded. D a t in rem ining col –9 –9 71 (2) 99 (1 3 ) 99 (12) 5. 111 (2) 97 (2) 159 ( 3 ) 3 40 (6) 90 (2) – 6.1 4 8 .4 6.1 3 15 (6) 99 (6) – 6 25.9 5.9 24.4 111 (2) 99 (14) 99 (11) 5.71 49 6 rk gill protein s h a rk gill 26 – – – 9. 3 3 1– ––6. 3 9 – – 92 (5) 6 25.9 5.9 24.4 38 338 – – 106 (2) – – – 41 (2) – 7. 8 –– –– 8 7 202 3 10 –91104 – – 3 M as cot M S BLA TP PEAK S 21 – – – 92 (2) – 6.2 46.2 6. 3 21 101 1 8 3 914 15 – – 10 8 (2) 38 for leop a rd s core for – – – – – 47 (2) 26 (2) 5.5 54.7 6.1 45.6 8 22 2––8 629– – 6 160 24 76 4 8 14 3 161 47 144 46 – – – 5. 127 24 11 8 11 8 742 69 696 6 8 251 3 51 4 3 P S D Cov. CID Cov. P S DP a tion No. Acce ss ion gi| 83 9 38 66 69 gi|14250 3 17 7 3 gi|5752 8 2 38 gi|41056095 72gi|47225709 6 – – 66 (1) 75 (2) – 5.6 2 gi| 338 60179 65gi|4624975 8 15 – – – 6 8 (2) –3 79 5. gi| 38 50 33 0 3 gi| 3 026 8 616 – – gi|46 8 4944 383 gi| 3 026 8 616 – – 112 19 – – – 6. 33 gi|947 3 2607 217 gi|41 38 674 3 gi| 38 1 8 888 gi|4720 83 57 – – – – – 70 (2) – 5 gi|4720 83 57 – – 172 24 – 11 (2) 99 (2) 5 gi|947 3 2607 gi|1490 3 4975 157 4 8 gi|14 8 7255 3 7 – – – – – 90 (2) 90 (1) 5.2 gi|1490 3 664 a lph ) ) (P S MC2) gi|19709 8 6 88 ) (P S MC2) gi|410557 38 8 27 64 597 4 8 s pectrometry identific ) (P S MD12) gi|62 8 5 83 99 – – – – – 7 8 (1) 4 8 (2) 5.9 51.4 6 4 3 .2 ) (P S MA2) gi|6677 3 056 – – 156 ) (P S MA2) gi|6677 3 056 ) (ALDOA) a nd m ass ) (EEF2) ) (DDAH1) ) (CPOX) ) (MVP) ) (TOP1) ) (ACTB) ) (OAT) ) (PPA1) ) (PPA1) s e a w ter ( S W)] ) (PMM2) ) (PEPD) ) (ANXA4) nic ( Danio rerio ) (PL S3 a ldol as e A ( Cephaloscyllium umbratile Protein identific a tion ( abb revi tion) ) (EZR) ) (P S MA2) ) (P S MA2) a n s l tion elong f ctor 2, like ( Danio rerio type 2 ( Danio rerio (P S MC2) type 2 ( Danio rerio u k a ryotic tr Pho s phom a nnom u t as e 2 ( Danio rerio Pl as tin 3 ( Homo sapiens Prote as ome (pro s ome, m a crop in) 26 S subu nit, ATP e 2 ( Danio rerio Ornithine a minotr n s fer as e ( Mus musculus t a tive f u nction bas ed on Gene Ontology nnot tion s nd m n ua l review of the liter re. Protein pot th were not identified re incl P 0.004 Bet a ctin (cytopl as mic) ( Rattus norvegicus 0.002 Peptid as e D ( Rattus norvegicus 0.007 Prote as ome (pro s ome, m a crop in) 26 S subu nit, ATP e 2 ( Pongo abelii 0.00 8 <0.001 Novel protein s imil a r to verte b te prote as ome (pro ome, m crop in) subu nit, <0.001 Prote as ome (pro s ome, m a crop in) 26 S subu nit, ATP e 2 ( Rattus norvegicus 3 2 <0.001 M a jor v au lt protein (Fr gment) ( Ictalurus punctatus tive to control 100 % 3 . Expre ss ion r a tio s [rel tive to 0.19 0.001 Pyropho s ph a t as e, inorg 3 .16 0.27 0.002 Glycer a ldehyde- 3 -pho s ph te dehydrogen as e ( Triakis scyllium ) (GAPDH) 3 .77 0.007 Glycer a ldehyde- 3 -pho s ph te dehydrogen as e ( Triakis scyllium ) (GAPDH) 0.45 0.022 Prote as ome 26 S subu nit, non-ATP e nit 12 ( Xenopus tropicalis 0. 3 1 0.00 3 50 % 40. Expre ss ion r a tio 45 0.4 8 33 2969 2.60 0.0 3 1 Dimethyl a rginine dimethyl minohydrol as e 1 ( Danio rerio 16 0. 38 44 0.2455 0.00 3 2.42 0.002 Coproporphyrinogen oxid as e ( Tetraodon nigroviridis 12 0.47 0.026 Ezrin ( Homo sapiens 15 0.29 0.00 8 2122 0.1 3 1410 0. 3 9 0.49 0.042 0.0 3 1E DNA topoi s omer as e I ( Chlorocebus aethiops 2 8 27 65 2.20 0.002 Novel protein s imil a r to verte b te prote as ome (pro ome, m crop in) subu nit, lph 42 0.17 0.001 Annexin A4 ( Rattus norvegicus 64 0.4 3 62 2.12 0.004 Prote as ome (pro s ome, m a crop in) subu nit, lph type, 2 ( Danio rerio 20 0.0 3 1 8 3 4 2.55 0.007 Pyropho s ph a t as e, inorg nic ( Danio rerio 66 0.46 0.005 Up s tre a m b inding tr n cription f ctor, RNA polymer as e I ( Mus musculus ) (UBTF) gi|12 3 241494 57 0. 3 4 0.004 Fr u cto s e- b i pho ph a te 61 0.22 0.002 Prote as ome (pro s ome, m a crop in) subu nit, lph type, 2 ( Danio rerio T ab le S pot (ii) Met ab oli s m of pho ph a te gro u p (vii) Amino a cid met ab oli s m nd u re cycle (ix) Glycoprotein b io s ynthe i (x) Heme b io s ynthe i (iv) V au lt s (v) Cyto s keleton (viii) Mem b r a ne inding Protein s were gro u ped b y p (vi) Prote as ome (iii) Tr a n s cription nd tr l tion (i) Glycoly s i

THE JOURNAL OF EXPERIMENTAL BIOLOGY Leopard shark proteomics 219

Table 4. Activities of Na+/K+-ATPase (mol ADP h–1 mg–1 protein) in leopard shark rectal gland, gill and kidney homogenates after salinity change in experiment 3 (24 h) and experiment 2 (3 weeks) Tissue Time 50–60% SW N 75–80% SW N 100% SW N Rectal gland 24 h 49.12±2.01a 6 63.98±3.83b 6 55.92±2.94a,b 5 Gill 24 h 0.30±0.14 6 0.30±0.09 6 0.27±0.06 5 3 wk 0.29±0.08 3 0.27±0.13 3 0.26±0.04 3 Kidney 24 h 1.09±0.11b,c 6 0.91±0.15b,c 6 0.70±0.03c 5 3 wk 2.07±0.04a 3 1.36±0.13b 3 1.09±0.16b,c 3 Values are means ± s.e.m. Lowercase superscripts indicate statistically different groups within each tissue, as determined by two-way ANOVA with time and salinity as factors. SW, seawater.

Table 5. Caspase 3/7 activity and chymotrypsin-like proteasome activity in leopard shark rectal gland and gill homogenates after salinity change in experiment 3 (24 h) and experiment 2 (3 weeks) Tissue Activity 50–60% SW N 75–80% SW N 100% SW N Rectal gland Caspase 3/7 24 h 1571±220 6 2611±633 6 2675±362 4 Gill Caspase 3/7 24 h 17,772±1795 6 13,968±3911 6 16,095±3863 5 Proteasome 24 h 396,133±26,329 6 344,223±45,015 6 381,978±64,287 5 Proteasome 3 weeks 386,277±86,447 3 431,432±26,730 3 301,548±71,377 3 Activities were determined by luminescent assays [relative light units (RLU)] and were normalized to total protein. Values are means ± s.e.m. (RLU mg–1 protein). There were no significant differences with salinity. SW, seawater.

100 a Leopard sharks ionoregulate while osmoconforming, A Swimming activity 24 hours 90 TBF 48 hours with a lag a,b Swimming activity 48 hours ) When faced with decreases in salinity, partially euryhaline –1 80 elasmobranchs such as the leopard shark eventually re-establish their 70 b slightly hyperosmotic status by selectively losing urea (and possibly 60 other plasma constituents not measured in the present study) while maintaining ion concentrations. Notably, the adjustments in plasma 50 osmolality, urea and ion concentrations in leopard sharks lagged 40 behind the changes in the medium, similar to patterns in other elasmobranchs (Cooper and Morris, 1998; Pillans et al., 2005). Some 30 of our physiological measurements indicate active regulation in 20 response to salinity change, while others are more indicative of passive movements of water and plasma constituents. The 10 50–60 75–80 100 hematological results revealed no effect of salinity on HCT or RBC 3.0 characteristics, as found in other elasmobranch studies (e.g. Janech 100 Swimming activity et al., 2006; Sulikowski et al., 2003). Similarly, plasma B TBF Number swimming concentrations of sodium, chloride and potassium ions either 2.5 remained unchanged or stabilized at new, lower levels by 48 h that b b 80 persisted over the 3 weeks. Interestingly, the fairly rapid decreases 2.0 in leopard shark rectal gland Na+/K+-ATPase activity and size within wimming 24 h are analogous to, but less severe than, those observed in rectal 60 s 1.5 glands of elasmobranchs acclimated to FW for longer periods (Piermarini and Evans, 2000; Pillans et al., 2005). These changes 40 are consistent with a reduced physiological capacity for NaCl 1.0 a excretion in dilute seawater, despite the persistent, but reduced, s min s troke S wimming a ctivity ( % of time) or t il- b e fre qu ency (TBF; inwardly directed Na+ and Cl– gradients in leopard sharks.

20 0.5 N u m b er of s h a rk Given that leopard sharks are very rarely captured near or below 50% SW, our data are consistent with the hypotheses that partially 0 0 euryhaline sharks cannot switch from net ion excretion to net ion 60 80 100 uptake and also cannot defend large hyperosmotic gradients. These Salinity (% SW) physiological limitations contrast with the abilities of truly euryhaline elasmobranchs in FW (Janech et al., 2006; Pillans and Fig. 5. Short-term (A, 24 h and 48 h) and long-term (B, 3 weeks) behavioral responses of leopard sharks to salinity change, as determined by focal Franklin, 2004; Pillans et al., 2005). It is possible that partially animal sampling and point sampling techniques. Different lowercase letters euryhaline elasmobranchs simply cannot regulate the abundances represent significant effects of salinity on two of the behavioral or activities of necessary epithelial transporters when facing dilute measurements. salinity. Plasma urea decreased in the long-term in leopard sharks

THE JOURNAL OF EXPERIMENTAL BIOLOGY 220 W. W. Dowd and others

more proteins were regulated in rectal gland by a more extreme physiological disturbance (i.e. more changes in 50% SW than in 75% SW).

Regulation of intracellular osmolytes and energy producing and consuming processes Gill and rectal gland exhibited several common responses after exposure to 50% SW, including changes in expression of proteins involved in the regulation of intracellular osmolyte pools. Both Fig. 6. Close-up of 2-D gel images from leopard shark gill 24 h after change to 100% seawater (SW) (controls, left) and 50% SW (right). Outlines tissues exhibited changes in the abundance of proteins associated indicate two pairs of protein spots, all four identified as proteasome alpha with GO annotations for amino acid metabolism (of which DDAH1 type 2 (left pair: 61, 62; right pair: 64, 65 in Table 3), that changed and OAT in gill were discussed above). Several amino acids are isoelectric points between the two treatments. important secondary osmolytes in elasmobranch osmoregulation, particularly intracellularly, and some (e.g. -alanine, proline) have been shown to decrease with decreasing salinity (Steele et al., 2005). exposed to 50–60% or 75–80% SW, driving the continued decreases In addition, both the rectal gland and gill exhibited changes in in plasma osmolality at 3 weeks. Given the evidence for decreased proteins involved in the inositol metabolism pathway (rectal gland: urea production in lower salinities for both euryhaline (Anderson ALDH6A1 F 50% SW; gill: ALDOA f). Myoinositol may play a et al., 2005) and partially euryhaline elasmobranchs (Steele et al., more prominent role in elasmobranch tissue osmoregulation than 2005), differences in the ability to retain urea probably contribute previously acknowledged (Steele et al., 2005; Yancey, 2001). The to differences in salinity tolerance between these two groups. increase in ALDH6A1 in rectal gland is consistent with increased Consistent with this idea and with our data, other partially euryhaline catabolism of -alanine and/or myoinositol following salinity species increase urea loss in dilute seawater (Steele et al., 2005). decrease in order to equilibrate intracellular osmolarity with that of Elasmobranch epithelial membrane permeabilities to urea are the plasma (and SW). Decreased abundance of PEPD in gill already quite low in SW (Zeidel et al., 2005; Wood et al., 1995) suggests reduced hydrolysis of proline-containing dipeptides and, at least in gill, are reinforced by active transport of urea back (Tanoue et al., 1990), thus inhibiting accumulation of intracellular into the plasma (Fines et al., 2001). Interestingly, our proteomics proline. Clearly further research on the interactions of intra- and data suggest decreased urea production at low salinity within gill extracellular osmolyte pools and the enzymes and transporters cells themselves. The decrease in OAT (all abbreviations as in regulating their abundance is warranted in the context of Tables 2 and 3) abundance implies reduced conversion of ornithine elasmobranch euryhalinity. to glutamate for entry into the urea cycle (Ballantyne, 1997). The Both tissues also exhibited overall decreases in proteins related increase in DDAH1, which catalyzes the conversion of to energy metabolism. Redirection of cellular energy supply is a dimethylarginine to dimethylamine and citrulline, also may classic component of the ‘cellular stress response’, during which reduce the availability of arginine for the urea cycle. Extrahepatic processes that protect from and repair macromolecular damage urea cycle activity in elasmobranchs has recently been increase at the expense of cell growth and proliferation (Kültz, 2003; demonstrated (Steele et al., 2005), and our data suggest that Kültz, 2005). However, our data are more consistent with reduced several elasmobranch tissues may adjust urea production energy/ATP requirements at lower salinity, particularly in rectal according to environmental conditions. gland. Our proteomics data suggest decreased glycolytic flux in both rectal gland (GAPDH f 75% SW, PGAM2 f 50% SW) and gill Proteomics reveals coordinated responses to salinity change (ALDOA f, opposite regulation of two GAPDH spots implying post- Given the changes we found in plasma composition, it follows that translational modifications). Further changes relevant to energy leopard shark cells, even those not exposed directly to the production were evident in rectal gland (TCA cycle, IDH2 f 50% environment, would be osmotically challenged by reduced water SW; malate aspartate shuttle, MDH1 f 50% SW; oxidative salinity. For example, decreases in total intracellular urea and TMAO phosphorylation, ATP5A1 mostly decreases 50% SW). The opposite concentrations and/or the ratio of these counteracting osmolytes may pattern of regulation of two gill spots identified as PPA1 could also have significant effects on shark enzyme structure and function indicate modulation of oxidative phosphorylation due to PPA1’s (Hochachka and Somero, 2002; Yancey and Somero, 1978). Our role in phosphate metabolism and ATP synthesis. Interestingly, there proteomics results and pathway modeling indicated a coordinated, were no significant changes in proteins directly associated with fuels fairly rapid (within 24 h) response of several functional processes other than glucose, despite the fact that ketones and/or fatty acids to salinity change in leopard shark tissues. Several of the identified may be important energy sources in elasmobranch tissues proteins (e.g. GAPDH, IDH2, TOP1) are represented in the highly (Ballantyne, 1997). conserved ‘minimal stress proteome’ (Kültz, 2003; Kültz, 2005), Our biochemical results indicate that decreased energy demand although they were largely downregulated here rather than being in rectal gland in 50% SW may be driven by reduced activity of induced, as would be expected under ‘stressful’ conditions. Similar the Na+/K+-ATPase pump, whereas energy use in gill is probably functions are influenced by decreasing salinity in the leopard shark not changing due to direct, organismal ionoregulatory functions. rectal gland as were regulated in response to salt loading after feeding Interestingly, abundance of the ribosomal translation elongation in the rectal gland of spiny dogfish (Squalus acanthias) (Dowd et factor EEF2 decreased significantly in leopard shark gill in 50% al., 2008), although largely in the direction of gland inactivation in SW. Two proteins involved in the transcription of ribosomal the current context. Exposure to hyposmotic conditions also RNAs also decreased in gill. Binding of the transcription factor influences similar proteins and biological processes to those involved UBTF to upstream promoters is necessary for ribosomal RNA in the hyperosmotic response of a euryhaline teleost (tilapia, expression (Grummt, 1999). TOP1 catalyzes the breaking and Oreochromis mossambicus) (Fiol et al., 2006). We also found that religation of bonds in the DNA backbone to relieve torsion caused

THE JOURNAL OF EXPERIMENTAL BIOLOGY Leopard shark proteomics 221 by transcription; it is concentrated in nucleoli, the sites of Cytoskeletal and membrane rearrangement within ribosomal DNA sequences (Leppard and Champoux, 2005). existing cells Given that ribosomal RNA translation constitutes 40–60% of Both gill and rectal gland exhibited consistent decreases in proteins translational activity (Hannan et al., 1998), these findings suggest associated with different components of the cytoskeleton. In rectal a decrease in an energetically expensive process in gill during gland these included three keratins (all sequenced in elasmobranchs), low salinity challenge. Similarly, in rectal gland we observed which may indicate changes in cellular intermediate filament downregulation of several heterogeneous nuclear structure associated with the decrease in gland size. The rectal gland ribonucleoproteins, which regulate post-transcriptional processing exhibits a surprising degree of morphological plasticity during gland and alternative splicing of mRNA (Martinez-Contreras et al., activation after feeding (Matey et al., 2009). We hypothesize that 2007), as well as PRPF4. These results possibly indicate a analogous, but inverse, morphological reorganization operates in decrease from constitutive levels of transcription and protein the rectal gland of leopard sharks exposed to decreasing salinity. synthesis in favor of stabilizing, repairing or degrading existing In gill, three proteins associated with the actin cytoskeleton macromolecules (Kültz, 2005). decreased in abundance. EZR is a membrane-actin cytoskeleton crosslinker that prefers binding to ACTB (also decreased in gill) Proteasomal degradation and apoptosis (Chen and Wagner, 2001). PLS3 belongs to a highly conserved Although our proteomics data suggested responses related to family of actin bundling proteins (Delanote et al., 2005). ANXA4 proteasomal degradation of ubiquitinated proteins and cell death, (decreased in gill) belongs to a family of calcium-dependent, especially in gill, our biochemical measurements of these processes phospholipid-binding proteins. It associates with the apical revealed no effect of salinity. The 26S proteasome is an extremely membrane in epithelial cells, forms rigid 2-D arrays that may limit large protein complex, and it is subject to complex regulatory the mobility of integral membrane proteins (Piljic and Schultz, networks (Adams, 2003; Ciechanover and Schwartz, 1998; 2006), and inhibits epithelial calcium-dependent Cl– secretion in Glickman and Raveh, 2005). Thus, although we found that several colon cells (Xie et al., 1996). Overall, our proteomic and biochemical components of the proteasome were regulated by low salinity in data implicate morphological changes within existing cells (possibly gill, it is possible that these changes were insufficient to alter activity mediated by the proteasome), rather than cell turnover via apoptosis, of the entire complex. In addition, our assay measured chymotrypsin- in the leopard shark gill’s response to salinity change. like activity but not the other two known proteolytic activities of the proteasome (caspase-like and trypsin-like). The non-ATPase How is the molecular response to salinity change PSMD12, which decreased in gill, is a component of the 19S coordinated? regulatory lid that is necessary for organization and proper Our proteomics analyses tended to identify effector proteins at the localization of the mature proteasome (Isono et al., 2007; Yen et periphery of the IPA interaction networks, rather than the central al., 2003). PSMC2 (two spots decreased, one increased) also forms sensor/initiator nodes, which tend to operate largely via post- part of the 19S regulatory subunit, where it unfolds and translational signaling mechanisms that do not require changes in deubiquitinates proteins prior to their degradation in the 20S their abundance (Kültz, 2001a; Kültz, 2001b; Kültz and Avila, proteasome core. We also observed a slight shift in the isoelectric 2001). Along these lines, our pathway model for leopard shark rectal points of two pairs of protein spots identified as proteasome subunit gland suggested a central role for 14-3-3 zeta (YWHAZ), a alpha type 2 on gill 2-D gels between 50% SW and 100% SW phosphoprotein adapter implicated in the adaptive osmoregulatory (Fig. 6). PSMA2 forms part of the 20S proteolytic core, where it response in teleost gill (Kohn et al., 2003; Kültz et al., 2001). We regulates entry and exit of substrates (Bajorek and Glickman, 2004). know of no work on the function of 14-3-3 proteins in Together, our biochemical and proteomic data implicate as yet un- elasmobranchs. identified post-translational modifications of these proteasome Another intriguing result of our analysis is the presence of components, consistent with modulation of overall proteasome tumor necrosis factor  (TNF) – a highly conserved, pleiotropic substrate specificity (Glickman and Raveh, 2005; Hartmann- cytokine which also exhibits paracrine and autocrine activities – Petersen and Gordon, 2004). The consequences of these salinity- as a central node in the protein interaction networks of both rectal induced modifications of proteasome constituents in leopard shark gland and gill. We hypothesize that TNF is a key modulator of gill remain to be determined. the osmoregulatory response in elasmobranch fishes. The effects We found no evidence of changes in caspase 3/7 activity with of TNF on ion and water balance may be mediated by salinity- salinity change in either tissue. We have also found no late apoptotic dependent changes in the activity of the calcium-sensing receptor DNA degradation signal in leopard shark gill using a TUNEL assay [implicated in elasmobranch salinity sensing (Nearing et al., (W.W.D., unpublished observations). These data differ from trends 2002)] and/or the angiotensin II system [a major player in found in tilapia transferred from FW to SW, which exhibit a rapid elasmobranch volume regulation (Anderson et al., 2006; Anderson (within 6 h) and sustained increase in several measurements of et al., 2007)], as has been demonstrated in the mammalian kidney apoptosis in gill (Kammerer and Kültz, 2009). Because partially (Abdullah et al., 2006; Abdullah et al., 2008; Escalante et al., euryhaline elasmobranchs appear to lack the tilapia’s ability to 1994; Ferreri et al., 1998; Ferreri et al., 1997). Binding of TNF remodel/turnover ionoregulatory epithelia to switch between ion ligand can trigger apoptosis, activate gene transcription, stimulate excretion and ion absorption, apoptosis would be counterproductive amino acid uptake and/or have prosurvival (i.e. anti-apoptotic) for the organism in the short-term when behavioral avoidance is effects (Dempsey et al., 2003; Inoue et al., 1995; Wajant et al., still possible and accumulated cellular damage is still relatively low. 2003). TNF is also known to suppress the vault constituent MVP Rather, leopard shark cells appear to be activating acclimatory (decreased here in gill) (Stein et al., 1997). TNF expression physiological mechanisms to solve the problems caused by low- increased in salmonid gills around the parr-smolt transformation salinity exposure. We know of no other data evaluating the role of (Ingerslev et al., 2006), although the authors of this study did not apoptosis in osmoregulatory organs of elasmobranchs facing salinity relate this change to osmoregulatory adjustments. TNF also change. factored in the hyperosmotic response network for tilapia gill cells

THE JOURNAL OF EXPERIMENTAL BIOLOGY 222 W. W. Dowd and others

(Fiol et al., 2006), but again no mechanism for its action was seawater conditions. Recently, Ubeda et al. showed that bonnetheads postulated. (Sphyrna tiburo) move out of low salinity areas within a few days after salinity decreases (Ubeda et al., 2009). Should we find that Do leopard sharks use behavior to offset or avoid leopard sharks occupy low and/or variable salinity conditions for physiological costs of salinity change in estuaries? extended periods in the wild, it would argue strongly for benefits In scenarios where an environmental challenge nears or exceeds such as food availability and predator avoidance outweighing the physiological plasticity, we would predict an important role for physiological costs of inhabiting dynamic estuarine habitats. behavior in mitigating or avoiding potential physiological costs (Breuner and Hahn, 2003; Kidder, 1997; Schreck et al., 1997). LIST OF ABBREVIATIONS ‘Behavioral osmoregulation’ in fishes could be driven by the BCA bicinchoninic acid energetic costs of osmoregulatory mechanisms (Kidder et al., 2006), CID collision-induced dissociation and recent tracking studies have provided support for salinity-related FW freshwater movements in estuarine elasmobranchs (Heupel and Simpfendorfer, GO gene ontology HCT hematocrit 2008; Ubeda et al., 2009). Bat Rays (Myliobatis californica) IPA Ingenuity Pathways Analysis exhibited short-term doubling in organismal metabolic rate with MCV mean corpuscular volume salinity decrease (Meloni et al., 2002), and the organismal metabolic Mr molecular mass rate of leopard sharks doubled 1–2 h after transfer from 100% SW MS mass spectrometry to 45% SW (C. Mull and J.C., Jr, unpublished observations). This pI isoelectric point energy may be more beneficially spent on moving out of the low- PSD post-source decay salinity conditions, especially if the longer-term consequences of RBC red blood cells exposure are even more severe. Even if leopard sharks suppress SW seawater TCA trichloroacetic acid apoptotic pathways (as suggested by our data, although we cannot TFA trifluoroacetic acid rule out apoptosis in other tissues or at other time points), the costs of coping with salinity change (e.g. via ATP-dependent proteasomal ACKNOWLEDGEMENTS degradation) could be significant enough to trigger behavioral We acknowledge the support of the many people at Bodega Marine Laboratory responses. Thus, we tentatively interpret the short-term increase in (BML) who shared their laboratories and expertise. Greg Soyster, Stacey Fong, and Michelle Dulake assisted with experiments and laboratory analyses. Bob leopard shark swimming activity at low salinity as an attempted Kaufman, Heather Watts, and William Jewell (UC Davis Campus Mass avoidance of physiologically challenging conditions. Long-term Spectrometry Facility) provided technical assistance and numerous suggestions. physiological costs could require sharks to reduce their activity when This work was supported by several grants to W.W.D.: NSF Doctoral Dissertation Improvement Grant IOS–0709556, a Coastal Environmental Quality Initiative avoidance is not possible (as seen here), employing an ‘evade, then Fellowship from the UC Marine Council, a Mildred E. Mathias Student Research survive’ strategy. Grant from the UC Reserve System, American Elasmobranch Society Student The maintenance of urea concentrations in dilute salinity in the Research Awards, Bodega Marine Laboratory Intercampus Travel Grants, UC Davis Jastro-Shields Research Fellowships, a Marin Rod and Gun Club short-term preserved the massive gradient between the shark and Scholarship, and a Stockton Sportsmen’s Club Research Scholarship. B.N.H. was the environment for urea loss while exacerbating the problem of supported by the BML NSF REU program (DBI-0453251). Additional support osmotic water gain, documented here by the increase in muscle came from UC Agriculture Experiment Station grant 3455H to J.J.C. and NSF moisture. To maintain extracellular volume in dilute SW, renal grant IOS-0542755 to D.K. glomerular filtration rate and urine flow rate increase manyfold in REFERENCES elasmobranchs (Cooper and Morris, 2004; Goldstein and Forster, Abdullah, H. I., Pedraza, P. L., Hao, S., Rodland, K. D., McGiff, J. 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